Initial efforts to design protein catalysts (enzymes) relied on the modification of individual proteins. (Kaiser 1984; Knowles 1987) Despite some successes, (Wharton 1985; Wilks 1988; Hilvert 1985, 1989, 1994; Imperiali 1994; Johnson 1993) protein engineering has proven to be difficult and has suggested that notions of how enzymes work may still be naive. Combinatorial techniques, which rely on generating and screening large pools of protein variants simultaneously, offer a promising new approach to enzyme design. (DeGrado 1997) Several straightforward methods exist for generating large libraries (>1012) of proteins rapidly. (Reidharr-Olson 1991; Eisenbeis 1985; Wells 1985; Zoller 1983; Leung 1989; Crameri 1998; Zhang 1997, 1999; Stemmer 1994(a); Stemmer 1994(b)) Methodologies for identifying protein catalysts from libraries of proteins primarily have been based not on catalysis, but on binding to a transition-state analog, (Wagner 1995, 1998; Shokat 1989) as in the case of catalytic antibodies (Schultz 1989; Schultz 1995; Hilvert 1985, 1989, 1994; Posner 1994) and phage-display (Baca 1997). While antibodies clearly can catalyze a broad range of reactions, there are few reports (Jacobsen 1992) that selections for binding can generate catalysts that rival natural enzymes. In vivo complementation of essential enzymes, such as chorismate mutase and triosephosphate isomerase, offers a direct selection for catalysis but is limited to existing reactions. (Hermes 1990; Kast 1996) General screens and selections for catalysis are beginning to be reported. (DeGrado 1997; Koltermann 1998; Pedersen 1998)
Combinatorial techniques allow structure-activity relationships of enzymes to be amassed quickly. With the aid of powerful selections it should be possible to create synthetically useful catalysts for pharmaceuticals and materials. However, as with proteins, it is difficult to design screens for non-protein catalysts.
Screens have been developed based on small-molecule inducible gene expression. Several systems for small-molecule inducible gene expression have been developed to the point that they are integral to basic research. The discovery that the lac operon is induced by binding of lactose to the lac repressor led to the widespread use of isopropyl-b-D-thiogalactoside (IPTG) to induce gene expression in bacteria. More recently it has been shown that by fusing the tet repressor to a eukaryotic transcription activation domain, gene expression in eukaryotes can be both negatively and positively regulated using tetracycline.(Gossen 1992, Gossen 1995). The demonstration that transgene expression can be regulated with tetracycline in transgenic mice highlights the utility of this system. In addition to the tetracycline-based system, ecdysone-, (No) estrogen-, (Braselman 1993) and progesterone-regulated systems (Wang 1994) have been reported.
An extension of these strategies resulted from studies of the mechanism of action of the immunosuppressants FK506 and rapamycin. (Rosen 1992) It was found that the biological activity of both compounds resulted from the fact that they each dimerize two proteins, FKBP12 and calcineurin or FKBP12 and FRAP, that otherwise do not interact. One portion of FK506 binds to FKBP12 and another to calcineurin. Based on this understanding, it was demonstrated that these molecules could be used to control protein oligomerization inside a cell.
Molecules such as FK506 are small molecule ‘dimerizers’ (sometimes referred to as chemical inducers of dimerization, CIDs) that activate the function of numerous proteins that regulate many important cellular processes. Dimerizers allow the functions of proteins to be explored even when small molecule ligands are unknown. A limited number of such reagents have been synthesized that control the function of a much larger number of proteins (expressed as fusions of proteins of interest linked to a small molecule-responsive dimerization domain). See, e.g. Austin 1994, Choi 1996, Crabtree 996, Diver 1997, Ho 1996, Holsinger 1995, Hung 1996, Klemm 1998, Liberles 1997, Pruschy 1994, Schreiber 1998, Spencer 1996, Spencer 1995, Spencer 1993, Stockwell 1998, and Yang 1998.
To generalize this approach, it was shown in 1993 that two FK506 molecules tethered via their C21-allyl groups could oligomerize proteins fused to FKBP12. Specifically, several FK506 dimers termed “FK1012s” were shown to oligomerize the cytoplasmic domain of T-cell receptors when these domains were fused to the FK506-binding protein FKBP12. Since this initial paper, there have been several important extensions of this work by Schreiber and coworkers. Belshaw et al. reported in 1996 that two different proteins could be dimerized by tethering FK506 to cyclosporin. In 1997 Diver and Schreiber demonstrated a two-step synthesis of an FK1012 molecule based on recent olefin metathesis chemistry.
While this work with FK506 establishes a powerful new approach for manipulating cellular function with small molecules, optimized chemical handles that are more convenient to work with than FK506 are critical for realizing the potential of this approach. FK506 (FIG. 5B) is cell permeable and has excellent pharmacokinetic properties—as evidenced by its widespread use as an immunosuppressant. FK506, however, is not an ideal chemical handle. FK506 is not available in large quantities, coupling via the C21 allyl group requires several chemical transformations including silyl protection of FK506, (Spencer 1993, 1995, 1996; Pruschy 1994) and FK506 is both acid and base sensitive.(Wagner 1998; Coleman 1989)
One very recent approach to replacing FK506 is to design synthetic ligands that also bind to FKBP12 with high affinity. In 1997 Amara at al. reported AP1510, a synthetic dimerizer that binds FKBP12 with high affinity and that can oligomerize proteins fused to FKBP12. Very recently a derivative of AP1510, “5S”, was prepared that binds with high affinity to a FKBP12 mutant. (Clackson 1998) This derivative is particularly interesting because it does not bind with high affinity to wild type FKBP12.
Recently a system has been reported, named the yeast three-hybrid system, for detecting ligand-receptor interactions in vivo. (Licitra, represented in FIG. 2; U.S. Pat. No. 5,928,868) This system is based on the principle that small ligand-receptor interactions underlie many fundamental processes in biology and form the basis for pharmacological intervention of human diseases in medicine. This system is adapted from the yeast two-hybrid system by adding a third synthetic hybrid ligand. The feasibility of this system was demonstrated using as the hybrid ligand a dimer of covalently linked dexamethasone and FK506. The system used yeast expressing fusion proteins consisting of a) hormone binding domain of the rat glucocorticoid receptor fused to the LexA DNA-binding domain and b) FKBP12 fused to a transcriptional activation domain. When the yeast was plated on medium containing the dexamethasone-FK506 heterodimer, the reporter genes were activated. The reporter gene activation is completely abrogated in a competitive manner by the presence of excess FK506. Using this system, a screen was performed of a Jurkat cDNA library fused to the transcriptional activation domain in yeast in the presence of a methasone-FK506 heterodimer. The yeast in this system expressed the hormone binding domain of rat glucocorticoid receptor/DNA binding domain fusion protein. Overlapping clones of human FKBP12 were isolated. These results demonstrate that the three-hybrid system can be used to discover receptors for small ligands and to screen for new ligands to known receptors.
Other approaches, which do not rely on a readout based on alterations in genetic expression, have also been developed. WO 96/30540 (Tsien et al.) discloses a screen for β-lactamase activity that uses fluorescence resonance energy transfer as the indicator of β-lactamase activity. The degree of fluorescence in this screen depends on the level of β-lactamase activity. Detection of β-lactamase activity relies on detection of changes in the degree of fluorescence.
However, it has not heretofore been suggested to use small molecule induced protein dimerization to screen for catalysis in vivo, and specifically, it has not been suggested to use an enzyme cleavable moiety to link two molecules to dimerize proteins.
This invention provides proteins de novo with prescribed binding and catalytic properties and permits screening cDNA libraries based on biochemical function. Being able to understand and manipulate protein-small molecule interactions has broad implications for basic biomedical research and the pharmaceutical industry. Proteins engineered to have unique binding or catalytic properties have already proven useful as biomedical reagents, medical diagnostics, and even therapeutics. As with site-directed mutagenesis before it, randomization and screening techniques also offer an entirely new approach to understanding the molecular basis for recognition and catalysis. Technically, a high-throughput approach such as that disclosed here would speed-up the research because the activity of thousands of protein variants can be measured simultaneously. Practically, we believe that powerful screens in combination with existing randomization techniques will make it possible to take an existing protein fold and “evolve” it into an enzyme with a new function generating useful catalysts for the pharmaceutical and chemical industries. Intellectually, the ability to modify substrate specificity and catalytic activity offers a new standard for “understanding” how enzymes function. A powerful screen is also critical to the end goal of genome sequencing efforts-determining the function of each and every protein, bypassing decades of detailed biochemical and genetic experiments to unravel complex biochemical pathways. Since the screen is done in vivo and in both prokaryotes and eukaryotes, the methodology can be applied to functional genomics and drug discovery. A cDNA library can be screened for all enzymes that form or cleave a specific type of bond. A library of small molecules can be screened for its ability to inhibit a specific enzyme. The screen selects for cell permeability, compatibility with the cellular milieu, and inhibition of enzyme activity. The key to all of these applications is a robust screen for enzymatic activity such as that disclosed here.